I would suggest doing some rigorous quality-control on your protein stock, e.g., mass-spec'ing. Also, it may help to aliquot small aliquots into pcr tubes and freeze at -80, and use only those stocks for crystallizations. I had exactly this problem, and finally concluded that the problem was heterogeneity of the protein stock.
JPK On Tue, Nov 23, 2010 at 11:24 AM, Joe <[email protected]> wrote: > Hi all, > I recently have problems reproducing some conditions identified from high > throughput screenings. > The initial screening (10 mg/ml protein, 0.5 ul well+ 0.6 ul protein, 23 C) > gave rise to at least three hits from different screen kits. > The follow-up grid optimization (1.5 ul well + 1.5 ul protein) varying > precipitant concentrations and pHs did not produce any crystals for all > three conditions. Instead, the drops have heavier precipitation > background. The following experiments have been done in order to get > crystals back. > 1. Seeding, with various precipitant concentrations > 2. Varying volume ratios between well solution and protein (from 2: 1 to 1: > 2 v/v). > 3. Using original solutions from screen kits. > 4. Hanging drops and sitting drops, > 5. Dispensing protein first or well solutions first. > 6. Using robot to set up drops on 96-well plate to see if I can reproduce > the original hits. > But none of them has worked so far. This is the first time I encountered > such a scale-up issue. I am running out of ideas, so hope you could give me > some suggestions. Thank you in advance. > -- > Best regards, > > Joe
